The objectives of this proposal are to elucidate the structural features that dictate the function of two pancreatic proteins indispensible for fat digestion, lipase and colipase. In particular, the amino acids of lipase that interact with lipids and with colipase, and that form the active site will be identified. Also, the amino acids involved in the binding of colipase to lipids and to lipase will be located. Spectral studies, chemical modification of specific amino acids, and the comparison of the primary structure of lipase and colipase to homologous proteins and to identical proteins from other species have provided evidence for the involvement of specific amino acids in the properties of lipase and colipase. The actual role for each of these amino acids will be tested by the production of mutant cDNAs that encode for proteins with systematically altered amino acids. These mutant proteins will be expressed in bacterial or mammalian cells and the purified proteins will be tested for lipid binding, binding to the other protein, and fat digestion. Fat malabsorption, resulting from the lipase and colipase deficiencies of exocrine pancreatic dysfunction, is associated with various diseases, including cystic fibrosis, chronic alcoholism, and premature newborns, that afflict a large segment of the population. A better understanding of the molecular basis for the interaction of lipase and colipase with each other and with fats and of the genetic elements governing the regulated expression of these proteins is crucial for a rational approach to treating patients with residual pancreatic function and in the design of more efficient pancreatic enzyme replacement therapy.